Leukotriene B4 (LTB4) is a potent pro-inflammatory activator of inflammatory cells, including neutrophils (J. Palmblad, J. Rheumatol. 1984, 13(2):163-172), eosinophils (A. M. Tager, et al., J. Exp. Med. 2000, 192(3):439-446), monocytes (N. Dugas et al., Immunol. 1996, 88(3):384-388), macrophages (L. Gagnon et al., Agents Actions 1989, 34(1-2):172-174), T cells (H. Morita et al., Biochem. Biophys. Res. Commun. 1999, 264(2):321-326) and B cells (B. Dugas et al., J. Immunol. 1990, 145(10):3405-3411). Immune cell priming and activation by LTB4 can promote chemotaxis, adhesion, free radical release, degranulation and cytokine release. LTB4 stimulates T-cell proliferation and cytokine release in response to IL-2, concanavalin-A and CD3 ligation (H. Morita et al., Biochem. Biophys. Res. Commun. 1999, 264(2):321-326). LTB4 is a chemoattractant for T-cells creating a functional link between early innate and late adaptive immune responses to inflammation (K. Goodarzi, et al., Nat. Immunol. 2003, 4:965-973; V. L. Ott, et al., Nat. Immunol. 2003, 4:974-981; A. M. Tager, et al., Nat. Immunol. 2003, 4:982-990). There is substantial evidence that LTB4 plays a significant role in the amplification of many inflammatory disease states (R. A. Lewis et al., N. Engl. J. Med. 1990, 323:645; W. R. Henderson, Ann. Intern. Med., 1994, 121:684), including asthma (D. A. Munafo et al., J. Clin. Invest. 1994, 93(3):1042-1050), inflammatory bowel disease (IBD) (P. Sharon and W. F. Stenson, Gastroenterology 1984, 86(3):453-460), chronic obstructive pulmonary disease (COPD) (P. J. Barnes, Respiration 2001, 68(5):441-448), arthritis (R. J. Griffiths et al., Proc. Natl. Acad. Sci. U.S.A. 1995, 92(2):517-521; F. Tsuji et al., Life Sci. 1998 64(3):L51-L56), psoriasis (K. Ikai, J. Dermatol. Sci. 1999, 21(3):135-146; Y. I. Zhu and M. J. Stiller, Skin Pharmacol. Appl. Skin Physiol. 2000, 13(5):235-245), and atherosclerosis (E. B. Friedrich, et al., Arterioscler. Thromb. Vasc. Biol. 2003, 23:1761-1767; K. Subbarao, et al., Arterioscler. Thromb. Vasc. Biol. 2004, 24:369-375; A. Helgadottir, et al., Nat. Genet. 2004, 36:233-239; V. R. Jala, et al., Trends in Immun. 2004, 25:315-322). LTB4 also simulates the production of various cytokines and may play a role in immunoregulation (A. W. Ford-Hutchinson, Immunology 1990, 10:1). Furthermore, it has been shown that LTB4 levels are elevated in brochoalveolar lavage fluid from patients with scleroderma lung disease (see Kowal-Bielecka, O. et al., Arthritis Rheum. (Nov. 30, 2005), Vol. 52, No. 12, pp. 3783-3791). Therefore, a therapeutic agent that inhibits the biosynthesis of LTB4 or the response of cells to LTB4 may be useful for the treatment of these inflammatory conditions.
The biosynthesis of LTB4 from arachidonic acid (AA) involves the action of three enzymes: phospholipase A2 (PLA2), to release AA from the membrane lipids; 5-lipoxygenase (5-LO), to form the unstable epoxide Leukotriene A4 (LTA4); and leukotriene A4 hydrolase (LTA4-h), to form LTB4 (A. W. Ford-Hutchinson, et al., Annu. Rev. Biochem. 1994, 63:383-347). The cysteinyl leukotrienes are formed by the addition of glutathione to LTA4 by the action of LTC4 synthase (Aharony, D., Am. J. Respir. Crit. Care Med. 1998, 157 (6, Pt 2), S214-S218) into the pro-inflammatory cysteinyl leukotrienes LTC4, LTD4 and LTE4. An alternative path for LTA4 is conversion via transcellular biosynthesis and the action of lipoxygenases into lipoxin A4 (LXA4) and lipoxin B4 (LXB4) (C. N. Serhan, Prostaglandins 1997, 53:107-137).
LTA4-h is a monomeric, soluble 69 kD zinc metalloenzyme. A high resolution crystal structure of recombinant LTA4-h with bound inhibitors has been obtained (M. M. Thunissen et al., Nat Struct. Biol. 2001, 8(2): 131-135). LTA4-h is a bifunctional zinc-dependent metalloenzyme of the M1 class of metallohydrolases. It catalyses two reactions: the stereospecific epoxide hydrolase reaction to convert LTA4 to LTB4 and a peptidase cleavage of chromogenic substrates. The Zn center is critical to both activities. LTA4-h is related to aminopeptidases M and B, which have no LTA4-hydrolase activity. LTA4-h has high substrate specificity, accepting only a 5,6-trans-epoxide with a free carboxylic acid at C-1 of the fatty acid. The double-bond geometry of the substrate is essential for catalysis. In contrast, LTA4-h peptidase activity appears to be promiscuous, cleaving nitroanilide and 2-naphthylamide derivatives of various amino acids, e.g. in particular alanine and arginine. Arg-Gly-Asp, Arg-Gly-Gly, and Arg-His-Phe tripeptides are hydrolyzed with specificity constants (kcat/Km) similar to the epoxide hydrolase reaction. There is no known physiological peptide substrate for LTA4-h.
LTA4-h is widely expressed as a soluble intracellular enzyme in intestine, spleen, lung and kidney. High activity levels are found in neutrophils, monocytes, lymphocytes and erythrocytes. Tissue macrophages can have high LTA4-h levels. An interesting feature is that the cellular distribution of LTA4-h and 5-LO are distinct, requiring close apposition of cells such as neutrophils and epithelial cells for efficient transcellular LTB4 synthesis. Many studies support this concept, including data from bone marrow chimeras derived from LTA4-h−/− and 5-LO−/− mice (J. E. Fabre et al., J. Clin. Invest. 2002, 109(10):1373-1380).
Studies have shown that introduction of exogenous LTB4 into normal tissues can induce inflammatory symptoms (R. D. R. Camp et al., Br. J. Pharmacol. 1983, 80(3):497-502; R. Camp et al., J. Invest. Dermatol. 1984, 82(2):202-204). Elevated levels of LTB4 have been observed in a number of inflammatory diseases including inflammatory bowel disease (IBD), chronic obstructed pulmonary disease (COPD), psoriasis, rheumatoid arthritis (RA), cystic fibrosis, multiple sclerosis (MS), and asthma (S. W. Crooks and R. S. Stockley, Int. J. Biochem. Cell Biol. 1998, 30(2):173-178). Therefore, reduction of LTB4 production by an inhibitor of LTA4-h activity would be predicted to have therapeutic potential in a wide range of diseases. This idea is supported by a study of LTA4-h-deficient mice that, while otherwise healthy, exhibited markedly decreased neutrophil influx in arachidonic acid-induced ear inflammation and zymosan-induced peritonitis models (R. S. Byrum et al., J. Immunol. 1999, 163(12):6810-68129). LTA4-h inhibitors have been shown to be effective anti-inflammatory agents in preclinical studies. For example, oral administration of LTA4-h inhibitor SC57461 caused inhibition of ionophore-induced LTB4 production in mouse blood ex vivo, and in rat peritoneum in vivo (J. K. Kachur et al., J. Pharm. Exp. Thr. 2002, 300(2): 583-587). Eight weeks of treatment with the same inhibitor significantly improved colitis symptoms in cotton top tamarins (T. D. Penning, Curr. Pharm. Des. 2001, 7(3):163-179). The spontaneous colitis that develops in these animals is very similar to human IBD. The results therefore indicate that LTA4-h inhibitors would have therapeutic utility in this and other human inflammatory diseases.
Events that elicit the inflammatory response include the formation of the pro-inflammatory mediator LTB4, which can be blocked with an LTA4-h inhibitor, thus providing the ability to prevent and/or treat leukotriene-mediated conditions, such as inflammation. LTA4-h inhibitors have been described, for example, in U.S. Pat. No. 7,737,145 and U.S. Patent Application Publication No. 20100210630A1, the contents of each of which are incorporated by reference herein.
It would be advantageous to develop additional LTA4-h inhibitors.